1. Technical Field
The presently disclosed invention embodiments relate to compositions and methods for the treatment of microbial infections. In particular, the present embodiments relate to improved treatments for managing bacterial infections in agricultural, industrial, manufacturing, clinical, personal healthcare, and other contexts, including treatment of bacterial biofilms and other conditions.
2. Description of the Related Art
The complex series of coordinated cellular and molecular interactions that contribute to responding to and resisting microbial infections and/or to healing or maintenance of plant and animal (including human) bodily tissues generally, may be adversely impacted by a variety of external factors, such as opportunistic and nosocomial infections (e.g., clinical regimens that can increase the risk of infection), local or systemic administration of antibiotics (which may influence cell growth, migration or other functions and can also select for antibiotic-resistant microbes), and/or other factors.
Unfortunately, systemically or locally introduced antibiotics are often not effective for the treatment of many chronic infections, and are generally not used unless an acute bacterial infection is present. Current approaches include administration or application of antibiotics, but such remedies may promote the advent of antibiotic-resistant bacterial strains and/or may be ineffective against bacterial biofilms. It therefore may become especially important to use antiseptics when drug resistant bacteria (e.g., methicillin resistant Staphylococcus aureus, or MRSA) are detected. There are many antiseptics widely in use, but bacterial populations or subpopulations that are established may not respond to these agents, or to any other currently available treatments. Additionally, a number of antiseptics may be toxic to host cells at the concentrations that may be needed to be effective against an established bacterial infection, and hence such antiseptics are unsuitable. This problem may be particularly acute in the case of efforts to clear infections from natural surfaces, including surface features on commercially and/or agriculturally important plants such as many crop plants, and also including internal epithelial surfaces, such as respiratory (e.g., airway, nasopharyngeal and laryngeal paths, tracheal, pulmonary, bronchi, bronchioles, alveoli, etc.) or gastrointestinal (e.g., buccal, esophageal, gastric, intestinal, rectal, anal, etc.) tracts, or other epithelial surfaces.
Particularly problematic are infections composed of bacterial biofilms, a relatively recently recognized organization of bacteria by which free, single-celled (“planktonic”) bacteria assemble by intercellular adhesion into organized, multi-cellular communities (biofilms) having markedly different patterns of behavior, gene expression, and susceptibility to environmental agents including antibiotics. Biofilms may deploy biological defense mechanisms not found in planktonic bacteria, which mechanisms can protect the biofilm community against antibiotics and host immune responses. Established biofilms can arrest the tissue-healing process.
Common microbiologic contaminants that underlie persistent and potentially deleterious infections include S. aureus, including MRSA (Methicillin Resistant Staphylococcus aureus), Enterococci, E. coli, P. aeruginosa, Streptococci, and Acinetobacter baumannii. Some of these organisms exhibit an ability to survive on non-nutritive clinical surfaces for months. S. aureus, has been shown to be viable for four weeks on dry glass, and for between three and six months on dried blood and cotton fibers (Domenico et al., 1999 Infect. Immun. 67:664-669). Both E. coli and P. aeruginosa have been shown to survive even longer than S. aureus on dried blood and cotton fibers (ibid).
Microbial biofilms are associated with substantially increased resistance to both disinfectants and antibiotics. Biofilm morphology results when bacteria and/or fungi attach to surfaces. This attachment triggers an altered transcription of genes, resulting in the secretion of a remarkably resilient and difficult to penetrate polysaccharide matrix, protecting the microbes. Biofilms are very resistant to the mammalian immune system, in addition to their very substantial resistance to antibiotics. Biofilms are very difficult to eradicate once they become established, so preventing biofilm formation is a very important clinical priority. Recent research has shown that open wounds can quickly become contaminated by biofilms. These microbial biofilms are thought to delay wound healing, and are very likely related to the establishment of serious wound infections.
Maintenance of intact, functioning skin and other epithelial tissues (e.g., generally avascular epithelial surfaces that form barriers between an organism and its external environment, such as those found in skin and also found in the linings of respiratory and gastrointestinal tracts, glandular tissues, etc.) is significant to the health and survival of humans and other animals.
Bismuth Thiol-(BT) Based Antiseptics
A number of natural products (e.g., antibiotics) and synthetic chemicals having antimicrobial, and in particular antibacterial, properties are known in the art and have been at least partially characterized by chemical structures and by antimicrobial effects, such as ability to kill microbes (“cidal” effects such as bacteriocidal properties), ability to halt or impair microbial growth (“static” effects such as bacteriostatic properties), or ability to interfere with microbial functions such as colonizing or infecting a site, bacterial secretion of exopolysaccharides and/or conversion from planktonic to biofilm populations or expansion of biofilm formation. Antibiotics, disinfectants, antiseptics and the like (including bismuth-thiol or BT compounds) are discussed, for example, in U.S. Pat. No. 6,582,719, including factors that influence the selection and use of such compositions, including, e.g., bacteriocidal or bacteriostatic potencies, effective concentrations, and risks of toxicity to host tissues.
Bismuth, a group V metal, is an element that (like silver) possesses antimicrobial properties. Bismuth by itself may not be therapeutically useful and may exhibit certain inappropriate properties, and so may instead be typically administered by means of delivery with a complexing agent, carrier, and/or other vehicle, the most common example of which is Pepto Bismol®, in which bismuth is combined (chelated) with subsalicylate. Previous research has determined that the combination of certain thiol-(—SH, sulfhydryl) containing compounds such as ethane dithiol with bismuth, to provide an exemplary bismuth thiol (BT) compound, improves the antimicrobial potency of bismuth, compared to other bismuth preparations currently available. There are many thiol compounds that may be used to produce BTs (disclosed, for example, in Domenico et al., 2001 Antimicrob. Agent. Chemotherap. 45(5):1417-1421, Domenico et al., 1997 Antimicrob. Agent. Chemother. 41(8):1697-1703, and in U.S. RE 37,793, U.S. Pat. No. 6,248,371, U.S. Pat. No. 6,086,921, and U.S. Pat. No. 6,380,248; see also, e.g., U.S. Pat. No. 6,582,719) and several of these preparations are able to inhibit biofilm formation.
BT compounds have proven activity against MRSA (methicillin resistant S. aureus), MRSE (methicillin resistant S. epidermidis), Mycobacterium tuberculosis, Mycobacterium avium, drug-resistant P. aeruginosa, enterotoxigenic E. coli, enterohemorrhagic E. coli, Klebsiella pneumoniae, Clostridium difficile, Heliobacter pylori, Legionella pneumophila, Enterococcus faecalis, Enterobacter cloacae, Salmonella typhimurium, Proteus vulgaris, Yersinia enterocolitica, Vibrio cholerae, and Shigella Flexneri (Domenico et al., 1997 Antimicrob. Agents Chemother. 41:1697-1703). There is also evidence of activity against cytomegalovirus, herpes simplex virus type 1 (HSV-1) and HSV-2, and yeasts and fungi, such as Candida albicans. BT roles have also been demonstrated in reducing bacterial pathogenicity, inhibiting or killing a broad spectrum of antibiotic-resistant microbes (gram-positive and gram-negative), preventing biofilm formation, preventing septic shock, treating sepsis, and increasing bacterial susceptibility to antibiotics to which they previously exhibited resistance (see, e.g., Domenico et al., 2001 Agents Chemother. 45:1417-1421; Domenico et al., 2000 Infect. Med. 17:123-127; Domenico et al., 2003 Res. Adv. In Antimicrob. Agents & Chemother. 3:79-85; Domenico et al., 1997 Antimicrob. Agents Chemother. 41(8):1697-1703; Domenico et al., 1999 Infect. Immun. 67:664-669: Huang et al. 1999 J Antimicrob. Chemother. 44:601-605; Veloira et al., 2003 J Antimicrob. Chemother. 52:915-919; Wu et al., 2002 Am J Respir Cell Mol Biol. 26:731-738).
Despite the availability of BT compounds for well over a decade, effective selection of appropriate BT compounds for particular infectious disease indications has remained an elusive goal, where behavior of a particular BT against a particular microorganism cannot be predicted, where synergistic activity of a particular BT and a particular antibiotic against a particular microorganism cannot be predicted, where BT effects in vitro may not always predict BT effects in vivo, and where BT effects against planktonic (single-cell) microbial populations may not be predictive of BT effects against microbial communities, such as bacteria organized into a biofilm. Additionally, limitations in solubility, tissue permeability, bioavailability, biodistribution and the like may in the cases of some BT compounds hinder the ability to deliver clinical benefit safely and effectively. The presently disclosed invention embodiments address these needs and offer other related advantages.
Protection of Plants and Agricultural Products: Description of the Related Art
In the agricultural and botanical arts there is a recognized need for formulations to reduce biofilms and disease in plants, and for methods of using such formulations on, e.g., seeds, plants, fruits and flowers, soil, and on cut flowers, trees, fruits, leaves, stems and other plant parts.
In agriculture, every year billions of dollars of crops are lost due to the formation of biofilms. The problem of anthracnose and biofilm-related diseases in plants is well known despite numerous unsatisfactory approaches that have attempted to address it. Plant diseases also affect industries involved in transporting and preserving fruit, vegetables, cut flowers and trees, and other plant products, as the normal protective mechanisms employed by intact living plants are no longer operative in the harvested product.
It is therefore desirable for agricultural purposes to reduce the amount of microbial growth on the surfaces of leaves, stems, fruits and flowers in situ, in transit or at commercial venues while maintaining compliance with environmental regulations. At the same time, it is desirable to allow for the flow of water within cut flowers, plants and trees to maintain plant tissue turgidity, integrity and quality in order to enhance the desirable characteristics of these products.
Organisms that cause infectious disease in plants include fungi, bacteria, viruses, protozoa, nematodes and parasitic plants. Insects and other pests also affect plant health by consumption of plant tissues, and by exposure of plant tissues to microbes.
Biofilms occur when bacteria bind to a surface, typically in an aqueous milieu such as under aquatic conditions or in water droplets or other conditions of high humidity, and after binding the biofilm formers begin to excrete a sticky substance which can then bind to a variety of materials including metals, plastics, medical implants and tissues. These biofilms can cause many problems, including degradation of materials and clogging of pipes, in industrial and agricultural environments, and infection of surrounding tissue when occurring in a medical environment. The medical field is particularly susceptible to problems caused by biofilm formation; implanted medical devices, catheters (urinary, venous, dialysis, cardiac) and slow-healing wounds are easily infiltrated by the bacteria present in biofilms. In agriculture, biofilms can cause mastitis, Pierce's disease, ring rot in potatoes, various crop blights and anthracnoses in many types of plants. Biofilms also reduce the quality and product life of cut flowers and trees.
Many plant diseases are caused by biofilm-producing bacteria indigenous to soil. Most microorganisms in the natural environment exist in multicellular aggregates generally described as biofilms. Cells adhere to surfaces and to each other through a complex matrix comprising a variety of extracellular polymeric substances (EPS) including exopolysaccharides, proteins and DNA. Plant-associated bacteria interact with host tissue surfaces during pathogenesis and symbiosis, and in commensal relationships. Observations of bacteria associated with plants increasingly reveal biofilm-type structures that vary from small clusters of cells to extensive biofilms. The surface properties of the plant tissue, nutrient and water availability, and the proclivities of the colonizing bacteria strongly influence the resulting biofilm structure (Ramey et al., 2004 Curr Opinion Microbiol. 7:602-9).
The terrestrial environment harbors abundant and diverse microbial populations that can compete for and modify resource pools. In this complex and competitive environment, plants offer protective oases of nutrient-rich tissues. Plants are colonized by bacteria on their leaves, roots, seeds and internal vasculature. Each tissue type has unique chemical and physical properties that represent challenges and opportunities for microbial colonists. Biofilms may form upon association or at later stages, with significant potential to direct or modulate the plant-microbe interaction. Additional temporal and spatial complexity arises as many microbes actively modify the colonized plant environment.
Surface-associated bacteria have a significant impact on agriculture. In developed countries, the losses caused by plant diseases reach up to 25% of crop yields, a percentage that is much higher in developing countries. Epiphytic populations constitute a reservoir and future source of infection, and can be found on host and non-host plants. Xylophylus ampelinus, a bacterial pathogen of grapevines, forms thick biofilms in the vasculature of these plants (Grail & Manceau 2003). Xylella fastidiosa is the causal agent of Pierce's disease in grapevines. X. fastidiosa is able to form biofilms within xylem vessels of many economically important crops. The mechanisms of pathogenicity are largely due to occlusion of xylem vessels by aggregation of X. fastidiosa and biofilm formation. Vessel blockage is believed to be a major contributor to disease development, with xylem sap providing a natural medium that facilitates the virulence of Pierce's disease of grapevine and citrus variegated chlorosis (Zaini et al., 2009 FEMS Microbiol LETT. 295:129-34).
One of the most relevant plant pathogens, Pseudomonas syringae, causes brown spot disease on bean. It colonizes the leaf surface sparsely in solitary small groups (fewer than ten cells), while larger populations (more than 1000 cells) primarily develop near trichomes or veins with higher nutrient availability. Large aggregates survive desiccation stress better than solitary cells. P. syringae survives as an epiphyte (i.e., colonizer of the aerial parts of plants) when not causing infections on host plant tissues (Monier et al. PNAS 2003; 100:15977-82).
Pseudomonas putida can respond rapidly to the presence of root exudates in soils, converging at root colonization sites and establishing stable biofilms (Espinosa-Urgel et al. Microbiol 2002; 148:341-3).
Xanthomonas campestris pv. campestris (Xcc) causes black rot on cruciferous plants, accessing the vasculature through wound sites in roots. Virulence involves degradative exoenzymes and the exopolysaccharide xanthan gum, which is necessary for virulence (Dow et al. PNAS 2003; 100:10995-1000).
Xanthomonas smithii subsp. citri is responsible for the disease, citrus canker. This disease has been found in most continents of the world except Europe. The pathogen has been eradicated in many countries. Xanthomonas smithii forms canker lesions on fruit, leaves and twigs of citrus plants. Wind-driven rain can spread the bacteria up to 15 km from the source to infect citrus trees via stomata or wounds (Sosnowski, et al. Plant Pathol 2009; 58:621-35).
Pantoea stewartii subsp. stewartii causes Stewart's wilt disease in maize and is transmitted by the corn flea beetle. The bacteria reside primarily in the host xylem and produce large amounts of exopolysaccharide (von Bodman et al. PNAS 1998; 95:7687-92).
Ralstonia solanacearum is a soil-borne pathogen that causes lethal wilt on many plants. Virulence depends on EPS and cell-wall-degrading enzymes controlled by a complex regulatory network (Kang et al. Mol Microbiol 2002; 46:427-37).
Clavibacter michiganensis subsp. sepedonicus is a Gram-positive phytopathogen that causes bacterial ring rot in potato. Marques and colleagues showed large bacterial, matrix-encased aggregates attached to the xylem vessels (Marques et al. Phytopathol 2003; 93:S57).
Biofilm-producing Erwinia chrysanthemi causes soft-rot disease through rapid maceration of plant tissue. The production of pectic enzymes may be quorum-sensing (QS)-regulated, and therefore the inability to form bacterial aggregates may preclude pectinolytic enzyme secretion. Erwinia amylovora, a related plant pathogen, infects approximately 75 different species of plants, all in the family Rosaceae. Hosts for this bacterium include apple, pear, blackberry, cotoneaster, crabapple, firethorn (Pyracantha), hawthorn, Japanese or flowering quince, mountain-ash, pear, quince, raspberry, serviceberry, and spiraea. The cultivated apple, pear, and quince are the most seriously affected species. A single fire blight epidemic in Michigan in 2000 resulted in the death of over 220,000 trees with a total loss of $42 million. Annual losses to fire blight and cost of control in the U.S. are estimated at over $100 million (Norelli et al. Plant Dis 2003; 87:26-32).
E. amylovora produces two exopolysaccharides, amylovoran and levan, which cause the characteristic fire blight wilting symptom in host plants (Koczan et al. Phytopathol 2009; 99:1237-44). In addition, other genes, and their encoded proteins, have been characterized as virulence factors of E. amylovora that encode enzymes facilitating sorbitol metabolism, proteolytic activity and iron harvesting (Oh & Beer. FEMS Microbiology Lett 2005; 253:185-192).
No matter which part of the plant is attacked by a microbial plant pathogen such as a biofilm-former, the effect is usually to weaken or kill the plant. By infecting the leaves, the pathogen compromises the plant's ability to produce its food (e.g., via photosynthesis). Some plant pathogens block the fluid transport vessels in the stems that supply the leaves, and when such pathogens attack the roots, the uptake of water and nutrients is reduced or stopped completely. Blockage of plant vasculature often involves biofilm-producing bacteria that clog the flow of water and nutrients, both in growing plants in soil and in cut plants in vase water.
When a plant is attacked by one of these microorganisms, the resulting damage provides an opportunity for additional microbial invasion of plant tissue and it is the combined onslaught that ultimately damages and destroys the plant. Plants that are under environmental stresses, such as drought or poor nutrition, are particularly e susceptible to microbial attack.
Sometimes the microbial ‘infection’ is symbiotic, where both organisms derive a benefit. A good example of this is the well known nitrogen fixing bacteria (Rhizobium) which reside in nodules on the roots of leguminous (pea family) plants—the plant provides food and protection, while the bacteria take nitrogen from the air and convert it to a form usable by the host. As another example, the Mycorrhizae are a whole Order of fungi that have a symbiotic relationship with plant roots. In view of such mutually beneficial symbioses, preservation or protection of plants against harmful microbial pathogens may desirably employ antimicrobial agents that do not disrupt these symbiotic relationships, wherever possible.
Saprophytic fungi are essential in breaking down dead organic matter to produce the humus which is needed for good soil structure. They do not have any chlorophyll and so cannot use light to capture energy (e.g., via photosynthesis); instead they derive their energy by breaking down plant and animal material—alive or dead. They can also live in a symbiotic relationship with certain plant species, e.g., the micorrhizae in the fine roots of conifers, which cannot survive without them to take up vital nutrients. The widespread use of chemical agents to control harmful plant pathogens can damage the balance of these beneficial fungi, and runs counter to the principals of organic management.
There are, however, other less welcome fungi, which attack living plants and weaken or kill them. Another category of microbial plant pathogens, viruses, may be resident within the cells of plant tissues and thus often cannot be treated with topically applied chemicals, such that affected plants must be destroyed. There are currently no antibiotics specifically developed for the treatment of plants (although some antibiotics developed for other purposes have found uses on plants), leaving a number of economically significant plant species vulnerable to pathogenic bacterial attacks. For instance, fireblight infestations of numerous plant species of the family Rosaceae have proven untreatable. Many harmful fungi, by contrast, can be killed with topically applied chemicals without damaging the plant host, because the fungal growth habitat is different, i.e., a number of undesirable pathogenic fungi tend to grow on plant surfaces and not within plant tissues, using root-like structures to extract nourishment.
Because killing many plant pathogens is often difficult or impossible, a number of strategies for protecting plants against deleterious microbial pathogens adopt the philosophy that “prevention is better than cure”. By observing good hygiene when propogating and growing plants, many microbial plant diseases can be prevented by blocking the opportunity for a microbial infection to be established. Often, significantly lower quantities of pesticides or microbicides can be effective when such agents are used prophylactically, rather than in response to an established infection.
Plants are also more susceptible to disease if they are not growing under optimal or near-optimal conditions, for example, due to poor soil quality (e.g., dearth of nutrients) by itself or in combination with drought or excessive rainfall or flooding. Extremely wet conditions can, for instance, promote pathogenic fungal and/or bacterial growth. Quorum sensing in P. syringae, for example, is dictated by water availability on the leaf surface (Dulla & Lindow. PNAS 2008; 105:3-082-7). Of course not all plant diseases can be prevented by good agricultural hygiene, insofar as some plant diseases are transmitted by insects and others are wind-borne. Aphids and other sap-sucking insects, for example, are the main vectors of viruses. Spores of fungal diseases are carried in the air, and in rain drops and splashes.
Biofilms on Seeds and Sprouts
Bacterial adherence to seeds is a process that strongly influences rhizosphere colonization. Seed suppliers often deliberately coat seed stocks with microbial biofilms to inoculate the developing rhizosphere. Conversely, biofilms on seeds and sprouts used for human consumption are often common sources of gastrointestinal infection. P. putida adheres effectively to seeds and will subsequently colonize the rhizosphere. Endophytic populations of nonpathogenic actinobacteria found in wheat tissues were derived from interior colonization by the actinobacteria of surface-sterilized seeds. Endophytic seed populations of beneficial nitrogen-fixing bacteria can help ensure future rhizosphere colonization. Other studies of seed colonization have reported rod shaped and coccal bacteria embedded within EPS in scanning electronmicrographs of alfalfa seeds and sprouts. Biofilms are notoriously resistant to washing and other common antibacterial treatments on seeds and sprouts. Fett et al. found that both Escherichia coli O157:H7 and Salmonella populations on alfalfa sprouts required treatments much harsher than simple water washing to reduce the numbers of adherent microbes, and full removal was never achieved. The surviving bacteria likely resided within biofilms (Ramey et al. Curr Opinion Microbiol 2004; 7:602-9).
Cut Flowers and Trees
Vascular pathogens inhabit the xylem or phloem of plant hosts and generally depend on insect vectors or wounding for dissemination. Cutting flowers or trees is a similar type of wounding that is especially prone to vascular infection. Biofilm bacteria enter and clog the vasculature at the cut surface, and interfere with the flow of water, minerals and nutrients. Cut flower preservatives diluted in vase water often contain salicylate or aspirin to reduce biofilm formation (Domenico et al., J Antimicrob Chemo 1991; 28:801-10; Salo et al., Infection 1995; 23:371-7), and provide a low pH to prevent bacterial growth and disrupt biofilms.
Antimicrobial Agents in Agriculture.
Eradication of plant pathogen incursions is very important for the protection of plant industries, managed gardens and natural environments worldwide. The consequence of a pathogen becoming endemic can be serious, in some cases having an impact on the national economy. The current strategy for eradication of a pathogen relies on techniques for the treatment, removal and disposal of affected host plants. There are many examples where these techniques have been successful but many where they have not. Success relies on a sound understanding of the biology and epidemiology of the pathogen and its interaction with the host. In examining examples of dealing with plant pathogens and diseased host material around the world, particularly Australasia, various techniques including burning, burying, pruning, composting, soil- and biofumigation, solarization, steam sterilization and biological vector control have been used (Sosnowski, et al. Plant Pathol 2009; 58:621-35).
Antibiotics have also been used since the 1950s, to control certain bacterial diseases of high-value fruit, vegetable, and ornamental plants. Today, the antibiotics most commonly used on plants are oxytetracycline and streptomycin. In the USA, antibiotics applied to plants account for less than 0.5% of total antibiotic use. Resistance of plant pathogens to oxytetracycline is rare, but the emergence of streptomycin-resistant strains of Erwinia amylovora, Pseudomonas spp., and Xanthomonas campestris has impeded the control of several important diseases. Thus, the role of antibiotic use on plants in the antibiotic-resistance crisis in human medicine is the subject of debate (McManus et al. Annu Rev Phytopathol 2002; 40:443-65).
The emergence of streptomycin-resistant (SmR) plant pathogens has complicated the control of bacterial diseases of plants. For example, in the United States, streptomycin is permitted on tomato and pepper for control of X. campestris pv. vesicatoria, but it is rarely used for this purpose because resistant strains are now widespread. Resistance in E. amylovora, the fire blight pathogen, has had widespread economic and political implications. Other phytopathogenic bacteria in which SmR has been reported include Pectobacterium carotovora, Pseudomonas chichorii, Pseudomonas lachrymans, Pseudomonas syringae pv. papulans, Pseudomonas syringae pv. syringae, and Xanthomonas dieffenbachiae (McManus et al. Annu Rev Phytopathol 2002; 40:443-65). The emergence SmR E. amylovora has intensified fire blight epidemics in the western USA and Michigan.
Streptomycin and oxytetracycline have been assigned the lowest toxicity category by the U.S. Environmental Protection Agency (EPA), and carcinogenic or mutagenic activities have not been observed for either antibiotic.
Alternatives to antibiotics are available and, at least to some extent, practical. Indeed, bacterial disease management in most cropping systems is based on the integration of genetic resistance of the host, sanitation (avoidance or removal of inoculum), and cultural practices that create an environment unfavorable for disease development. Biocontrol of plants using various species of bacteria and fungi is of growing interest. Rhizobacteria are considered as efficient microbial competitors in the root zone. Representatives of many different bacterial genera have been introduced into soils, onto seeds, roots, tubers or other planting materials to improve crop growth. These bacterial genera include Acinetobacter, Agrobacterium, Arthrobacter, Azospirillum, Bacillus, Bradyrhizobium, Frankia, Pseudomonas, Rhizobium, Serratia, Thiobacillus, and many others. Certain species of Bacillus, for example, can induce systemic resistance in many plants (Choudhary & Johri. Microbiol Res 2009; 164:493-513).
Application of copper compounds is effective in reducing populations of some bacterial plant pathogens, although several species have become resistant to copper (Cooksey Annu Rev Phytopathol 1990; 28:201-14), and most tree-fruit crops are sensitive to copper injury.
A number of synthetic and natural remedies exist for various plant diseases. Natural remedies include apple cider vinegar for leafspot, mildew and scab; baking soda spray for anthracnose, early tomato blight, leaf blight, powdery mildew and as a general fungicide; neem oil; sulfur; garlic; hydrogen peroxide; compost teas, etc. Numerous synthetic chemicals are used to prevent or treat plant disease, and come in water-soluble or water-insoluble formulations. Microbicides include phenoxarsine or a phenarsazine, maleimide, isoindole dicarboximide, halogenated aryl alkanol, 4-thioxopyrimidine derivatives (U.S. Pat. No. 6,384,040), heterocyclic organosiylyl compounds and isothiazolinone. Many microbicides are combined with pyrithione derivatives to make synergistic compounds (e.g., EP1468607). Certain isothiazolecarboxamides can be employed for the control of plant pests (e.g., U.S. Pat. No. 6,552,056; WO 2001/064644)
Recognizing the toxicity problem of microbicides in powder or crystalline form, U.S. Pat. No. Re. 29,409 teaches dissolving microbicides in liquid solvents, which may be added to the formulation mixture from which the end-use resin compositions are fabricated. Although liquid dispersions may be safely used at the site of preparing end-use resin compositions, careless use or disposal of the liquids may still pose environmental and health hazards. Alternatively, microbicides can also be administered in water-soluble thermoplastic resins. Microbicides can be added to rigid thermoplastic resin compositions and impart biocidal activity thereto so as to inhibit microbial growth on the surfaces thereof (U.S. Pat. No. 5,229,124). This is a solid, melt-blended solution consisting essentially of a microbicide dissolved in a carrier resin that is a copolymer of vinyl alcohol and (alkyleneoxy) acrylate. Although a microbicide may be a highly toxic chemical, its low concentration in the end-use product and its retention by the resin composition ensures that the microbicide in the end-use product poses no hazard to humans or animals.
Isothiazolinones are often used as microbicides in agriculture, for example, N-alkylbenzenesulfonylcarbamoyl-5-chloroisothiazole derivatives (e.g., U.S. Pat. No. 5,045,555). This microbicide is widely useful in, for example, the paper industry, textile industry, for producing coatings and adhesives, in painting, metal processing, in the resin industry, wood industry, construction industry, agriculture, forestry, fisheries, food industry and petroleum industry as well as in medicine. It exhibits an intense microbicidal effect, and can be added, in an appropriate amount, to processing water, circulating water, a raw material or a product. Further, it may be employed for disinfecting or sterilizing facilities, plants, livestock barns or instruments as well as seeds, seedlings and raw materials. Other derivatives of isothiazolone are also known (U.S. Pat. No. 3,523,121 and J. Heterocyclic Chem., 8, 587 (1971)). However, every known derivative compound is highly toxic to animals and fishes, which significantly restricts their application.
Sodium bicarbonate commonly has also been found to possess fungicidal properties when applied to plants, but typically requires frequent reapplication in order to realize efficacy.
The role of iron in plant host-parasite relationships has been elucidated in diseases as different as the soft rot and fire blight incited by Erwinia chrysanthemi and E. amylovora, respectively (Expert. Annu Rev Phytopathol 1999; 37:307-34). Because of its unique position in biological systems, iron controls the activities of plant pathogens. The production of siderophores by pathogens not only represents a powerful strategy to acquire iron from host tissues but may also act as a protective agent against iron toxicity. The need of the host to bind and possibly sequester the metal during pathogenesis is another central issue. Antimicrobials that interfere with bacterial iron uptake and cell respiration may play an important role in plant disinfection.
Many natural products (e.g., antibiotics) and synthetic chemicals with antimicrobial, antiseptic and in particular antibacterial, properties are known and have been at least partially characterized chemically and biologically. Exemplary characteristics include the ability to kill microbes (bactericidal effects), ability to halt or impair microbial growth (bacteriostatic effects), or ability to interfere with microbial functions such as colonizing or infecting a site, bacterial secretion of metabolites (some of which are malodorous), and/or conversion from planktonic to biofilm populations or expansion of biofilm formation (anti-biofilm effects). Antibiotics, disinfectants, antiseptics and the like (including bismuth-thiol or BT compounds) are discussed in U.S. Pat. No. 6,582,719, including factors that influence the selection and use of such compositions, including, e.g., bactericidal, bacteriostatic, or anti-biofilm potencies, effective concentrations, and risks of toxicity to host tissues.
Bacterial microcolonies protected within the biofilm are typically resistant to antiseptics or disinfectants. Antibiotic doses that kill free-floating bacteria, for example, need to be increased as much as 1,500 times to kill biofilm bacteria. At this high concentration, some antimicrobials can be toxic. Oxidizing brominated and chlorinated compounds, for example, are highly toxic and corrosive.
Suppression of the blossom-blight phase is a key to the management of fire blight. For blossom infection to occur, Erwinia amylovora to needs proliferate on stigmatic surfaces in an epiphytic phase. Rain is necessary for infection because it dilutes sugars on the hypanthium to osmotic potentials not inhibitory to E. amylovora. Rain is also important as an agent for redistribution of the bacterium from the stigmas to the hypanthium. These observations suggest that the optimal timing for use of antibiotic sprays is during this epiphytic phase, and after excessive rain (Johnson & Stockwell. Annu Rev Phytopathol 1998; 36:227-48).
Other bacterial epiphytes also colonize stigmas where they can interact with and suppress epiphytic growth of the pathogen. A commercially available bacterial antagonist of E. amylovora (BlightBan, Pseudomonas fluorescens A506) can be included in antibiotic spray programs. Integration of bacterial antagonists with chemical methods suppresses populations of the pathogen and concomitantly, fills the ecological niche provided by the stigma with a nonpathogenic, competing microorganism (Johnson & Stockwell. Annu Rev Phytopathol 1998; 36:227-48).
Pyrithione is the conjugate base derived from 2-mercaptopyridine-N-oxide (CAS#1121-31-9), a derivative of pyridine-N-oxide. Its antifungal effect resides in its ability to disrupt membrane transport by blocking the proton pump that energizes the transport mechanism. Experiments have suggested that fungi are capable of inactivating pyrithione in low concentrations (Chandler & Segel. Antimicrob. Agents Chemother 1978; 14:60-8). Zinc pyrithione is a coordination complex of zinc. This colorless solid is used as an antifungal and antibacterial agent. Due to its low solubility in water (8 ppm at neutral pH), zinc pyrithione is suitable for use in outdoor paints, cements and other products that provide protection against mildew and algae. It is an effective algaecide. It is chemically incompatible, however, with paints that rely on metal carboxylate curing agents. When used in latex paints comprising water that contains high amount of iron, a sequestering agent that will preferentially bind the iron ions is needed.
Particularly problematic in agriculture are infections composed of bacterial biofilms, a relatively recently recognized organization of bacteria by which free, single-celled (“planktonic”) bacteria assemble by intercellular adhesion into organized, multi-cellular communities (biofilms) having markedly different patterns of behavior, gene expression, and susceptibility to environmental agents including antibiotics. Biofilms may deploy biological defense mechanisms not found in planktonic bacteria, which mechanisms can protect the biofilm community against antibiotics and host immune responses. Established biofilms can arrest growth, development or wound-healing processes in plants.
Microbial biofilms are associated with substantially increased resistance to both disinfectants and antibiotics. Biofilm morphology results when bacteria and/or fungi attach to surfaces. This attachment triggers an altered transcription of genes, resulting in the secretion of a remarkably resilient and difficult to penetrate polysaccharide matrix, protecting the microbes. Biofilms are very resistant to the plant immune defense mechanisms, in addition to their very substantial resistance to antibiotics. Biofilms are very difficult to eradicate once they become established, so preventing biofilm formation is a very important agricultural priority. Recent research has shown that open wounds can quickly become contaminated by biofilms. These microbial biofilms are thought to impair growth, development and/or wound healing, and are very likely related to the establishment of serious and often intractable infections.
Clearly there is a need for improved compositions and methods for treating and preventing microbial infections in and on plants, including microbial infections that occur as biofilms. Certain embodiments described herein address this need and provide other related advantages.